Plasminogen activators contribute to age-dependent impairment of NMDA cerebrovasodilation after brain injury

Developmental Brain Research 156 (2005) 139 – 146 www.elsevier.com/locate/devbrainres

Research report

Plasminogen activators contribute to age-dependent impairment of NMDA cerebrovasodilation after brain injury William M. Armsteada,b,T, Douglas B. Cinesc,d, Abd Al-Roof Higazic,d,e a Department of Anesthesia, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA c Department of Pathology, University of Pennsylvania, Philadelphia, PA 19104, USA d Department of Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA e Department of Clinical Biochemistry, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel b

Accepted 10 February 2005 Available online 21 April 2005

Abstract Previous studies have observed that fluid percussion brain injury (FPI) impaired NMDA induced pial artery dilation in an age-dependent manner. This study was designed to investigate the contribution of plasminogen activators to impaired NMDA dilation after FPI in newborn and juvenile pigs equipped with a closed cranial window. In the newborn pig, NMDA (10 8, 10 6 M) induced pial artery dilation was reversed to vasoconstriction following FPI, but pretreatment with the plasminogen activator inhibitor PAI-1 derived hexapeptide (EEIIMD) (10 7 M) prevented post injury vasoconstriction (9 F 1 and 16 F 1, vs. 6 F 2 and 11 F 3, vs. 5 F 1 and 9 F 1% for responses to NMDA 10 8, 10 6 M prior to FPI, after FPI, and after FPI in EEIIMD pretreated animals, respectively). In contrast, in the juvenile pig, NMDA dilation was only attenuated following FPI and EEIIMD pretreatment partially prevented such inhibition (9 F 1 and 16 F 1 vs. 2 F 1 and 4 F 1 vs. 5 F 1 and 7 F 1% for responses to NMDA prior to FPI, after FPI, and after FPI in EEIIMD pretreated animals, respectively). Additionally, EEIIMD blunted age-dependent pial artery vasoconstriction following FPI. EEIIMD blocked dilation to the plasminogen activator agonists uPA and tPA while responses to SNP and papaverine were unchanged. Pretreatment with suPAR, which blocked dilation to uPA, elicited effects on pial artery diameter and NMDA vascular activity post FPI similar to that observed with EEIIMD. These data show that EEIIMD and suPAR partially prevented FPI induced alterations in NMDA dilation and reductions in pial artery diameter. EEIIMD and suPAR are efficacious and selective inhibitors of plasminogen activator induced dilation. These data suggest that plasminogen activators contribute to age-dependent impairment of NMDA induced dilation following FPI. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Newborn; Cerebral circulation; Excitatory amino acids; Plasminogen activators; Brain injury

1. Introduction Traumatic injury is the leading cause of death for infants and children and mortality is greatly increased in the presence of head injury [35]. While the effects of brain T Corresponding author. Department of Anesthesia, University of Pennsylvania, 305 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104, USA. Fax: +1 215 349 5078. E-mail address: [email protected] (W.M. Armstead). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.02.012

injury have been extensively investigated in adult animal models [28,40], less is known about brain injury in the newborn/infant. Cerebral blood flow falls and pial arteries constrict more in newborn (1–5 day old) versus juvenile (3– 4 week old) pigs following fluid percussion brain injury (FPI), a model of concussive head injury [19], supporting the idea that the newborn is more cerebral hemodynamically sensitive to brain injury [3]. Glutamate is an important excitatory amino acid transmitter in the brain. It can bind to any of three different

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ionotropic receptor subtypes named after specific synthetic analogues: N-methyl-d-aspartate (NMDA), kainate, and AMPA. Activation of NMDA receptors has been observed to elicit cerebrovasodilation and may represent one of the mechanisms for the coupling of local metabolism to blood flow [18]. All glutamate receptor subtypes have been implicated in neurotoxicity to some degree. However, the NMDA subtype is thought to play a crucial role in excitotoxic neuronal cell death [13]. Glutamatergic system hyperactivity has been demonstrated in animal models of traumatic brain injury, while NMDA receptor antagonists have been shown to protect against experimental brain injury [23,27,29]. However, while cerebral hemodynamics post insult is thought to correlate with neurologic status, little attention has been given to the role of NMDA vascular activity in the sequelae of traumatic brain injury. Recently, it has been observed that NMDA dilation is impaired after FPI in an age-dependent manner [4]. However, the mechanism for such impairment remains uncertain. Urokinase plasminogen activator (uPA) and tissue type plasminogen activator (tPA) are serine proteases that convert plasminogen to the active protease plasmin [11,15]. Urokinase is synthesized as a single chain (scuPA) zymogen that is converted to an enzymatically active two chain molecule (tcuPA) primarily through the actions of plasmin [34] or by binding to its cellular receptor urokinase plasminogen activator receptor (uPAR) [21]. Relatively little is known about the expression of uPA in the brain. In contrast, it has been shown that during neonatal development tPA mRNA levels are high in the brain due to involvement with neuronal migration and axonal growth [30]. Interestingly, tPA has also been observed to contribute to excitotoxic neuronal death by the activation of microglia [36,37] or enhancement of NMDA receptor mediated signaling [33]. Similarly, tPA protein was abundantly expressed after spinal cord injury while there was preservation of white matter and hindlimb motor function in tPAdeficient mice [1]. Alternatively, a neuroprotective effect of tPA in zinc induced neuronal death has also been documented [24]. More recent data show that the tPA induced opening of the blood–brain barrier following ischemia is NMDA receptor independent [41]. However, the role of tPA or uPA in the observed impaired dilation to NMDA receptor activation is unknown. This study investigated the contribution of plasminogen activators to age-dependent impairment of dilation to NMDA receptor activation. Soluble urokinase plasminogen activator receptor (suPAR) and plasminogen activator inhibitor (PAI-1) and a PAI-1 derived hexapeptide (EEIIMD) are purported inhibitors that have suggested selectivity for uPA and for both tPA and uPA, respectively [20,31,32]. Therefore, the study design included the determination of responses to NMDA receptor activation before and after FPI in the absence and presence of the administration of suPAR and the PAI-1 derived peptide EEIIMD.

2. Methods Newborn and juvenile pigs (1–5 days old and 3–4 weeks old, 1.2–1.6 and 6.1–8.0 kg respectively) of either sex were used in these experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Animals were sedated with isoflurane (1–2 MAC). Anesthesia was maintained with a-chloralose (30–50 mg/kg, supplemented with 5 mg/kg/h i.v.). A catheter was inserted into a femoral artery to monitor blood pressure and to sample for blood gas tensions and pH. Drugs to maintain anesthesia were administered through a second catheter placed in a femoral vein. The trachea was cannulated, and the animals were mechanically ventilated with room air. A heating pad was used to maintain the animals at 37–39 8C, monitored rectally. A cranial window was placed in the parietal skull of these anesthetized animals. This window consisted of three parts: a stainless steel ring, a circular glass coverslip, and three ports consisting of 17-gurage hypodermic needles attached to three precut holes in the stainless steel ring. For placement, the dura was cut and retracted over the cut bone edge. The cranial window was placed in the opening and cemented in place with dental acrylic. The volume under the window was filled with a solution, similar to CSF, of the following composition (in mM): 3.0 KCl, 1.5 MgCl2, 1.5 CaCl2, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO3. This artificial CSF was warmed to 37 8C and had the following chemistry: pH 7.33, pCO2 46 mm Hg, and pO2 43 mm Hg, which was similar to that of endogenous CSF. Pial arterial vessels were observed with a dissecting microscope, a television camera mounted on the microscope, and a video output screen. Vascular diameter was measured with a video microscaler. Methods for brain FPI have been described previously [37]. A device designed by the Medical College of Virginia was used. A small opening was made in the parietal skull contralateral to the cranial window. A metal shaft was sealed into the opening on top of intact dura. This shaft was connected to the transducer housing, which was in turn connected to the fluid percussion device. The device itself consisted of an acrylic plastic cylindrical reservoir 60 cm long, 4.5 cm in diameter, and 0.5 cm thick. One end of the device was connected to the transducer housing, whereas the other end had an acrylic plastic piston mounted on Orings. The exposed end of the piston was covered with a rubber pad. The entire system was filled with 0.9% saline. The percussion device was supported by two brackets mounted on a platform. FPI was induced by striking the piston with a 4.8 kg pendulum. The intensity of the injury (usually 1.9–2.3 atm, with a constant duration of 19–23 ms) was controlled by varying the height from which the pendulum was allowed to fall. The pressure pulse of the injury was recorded on a storage oscilloscope triggered photoelectrically by the fall of the pendulum. The amplitude of the pressure pulse was used to determine the intensity of the injury.

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2.1. Protocol Two types of pial vessels, small arteries (resting diameter, 120–160 Am) and arterioles (resting diameter, 50–70 Am), were examined to determine whether segmental differences in the effects of FPI could be identified. Typically, 2–3 ml of CSF was flushed through the window over a 30 s period, and excess CSF was allowed to run off through one of the needle ports. For sample collection, 300 Al of the total cranial window volume of 500 Al was collected by slowly infusing CSF into one side of the window and allowing the CSF to drip freely into a collection tube on the opposite side. Eight types of experiments were performed (all n = 6): (1) sham control, (2) sham treated with EEIIMD, (3) sham treated with suPAR, (4) FPI, (5) FPI pretreated with EEIIMD, (6) FPI pretreated with suPAR, (7) agonist selectivity responses in the absence and presence of EEIIMD, and (8) agonist selectivity responses in the absence and presence of suPAR. In experiments designed to investigate the influence of FPI on responses to excitatory amino acids, NMDA and glutamate (10 8, 10 6 M) were topically applied before and 60 min after FPI. EEIIMD or suPAR (10 7 M) were applied 30 min prior to FPI and responses after FPI obtained in the presence of continued administration of these agents. Sham control experiments were designed to obtain responses to excitatory amino acids and agonist selectivity agents initially (time 1) and then again 60 min later (time 2) (Table 1). In sham animals treated with EEIIMD or suPAR, responses to excitatory amino acids were obtained before and after either EEIIMD or suPAR. In the agonist selectivity experiments, responses to uPA and tPA (10 9, 10 7 M), sodium nitroprusside (SNP), and papaverine (10 8, 10 6 M) were obtained Table 1 Influence of NMDA, glutamate, tPA, and uPA on pial artery diameter Time 1 Small artery

Small artery

Arteriole

56 F 3 64 F 3T 69 F 3T

125 F 4 136 F 4T 144 F 5T

56 F 3 65 F 3T 70 F 3T

64 F 2 74 F 3T 81 F 3T

129 F 4 140 F 4T 149 F 5T

61 F 2 71 F 3T 78 F 3T

tPA ( log M) 0 130 F 4 9 139 F 4T 7 143 F 5T

52 F 1 56 F 2T 59 F 2T

129 F 3 137 F 4T 143 F 4T

52 F 1 56 F 2T 59 F 2T

uPA ( log M) 0 131 F 4 9 140 F 4T 7 146 F 4T

52 F 2 57 F 2T 61 F 2T

131 F 3 140 F 4T 145 F 4T

52 F 1 57 F 2T 61 F 2T

( log M) 122 F 4 134 F 4T 141 F 4T

Glutamate ( 0 120 8 132 6 139

log M) F4 F 3T F 4T

initially and then again 30 min after administration of either EEIIMD or suPAR. Methods previously published were used to make suPAR [10]. The vehicle for all agents was 0.9% saline. Because baseline pial vessel diameter changed as a result of the FPI intervention, data were calculated as the percent change from the baseline to normalize such differences. 2.2. CSF plasminogen activator determination Commercially available ELISA kits (Diapharma) were used to quantity CSF tPA concentration. In the first step, the CSF is introduced into a microwell coated with a highly purified monoclonal antibody specific for the tPA. tPA, if present in the sample, will bind to the solid phase antibody. Subsequently, a second non-competing anti-tPA antibody, labeled with horseradish peroxidase is added. With a positive reaction, this labeled antibody becomes bound to any solid-phase antibody/tPA complex previously formed. Incubation with enzyme substrate produces a blue color. The amount of color developed is directly proportional to the concentration of tPA in the tested sample. Total tPA is measured, free and tPA complexed with PAI-1. 2.3. Statistical analysis Pial artery diameter, CSF tPA, and systemic arterial pressure values were analyzed using ANOVA for repeated measures. If the value was significant, the data were then analyzed by Fishers protected least significant difference test. An a level of P b 0.05 was considered significant in all statistical tests. Values are represented as mean F SEM of the absolute value or as percentage changes from control values.

3. Results

Time 2 Arteriole

NMDA 0 8 6

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Values are Am F SEM, n = 6. T P b 0.05 versus corresponding control value (0).

3.1. Age-dependent influence of FPI on CSF tPA concentration CSF tPA concentration determined by ELISA was increased within 60 min of FPI (Fig. 1). The CSF concentration was significantly more robustly increased in newborn versus juvenile pigs (Fig. 1). On a molar basis, the baseline (pre-FPI) CSF concentration was approximately 10 10–10 9 M while the newborn FPI CSF concentration was approximately 10 8–10 7 M. 3.2. uPA and tPA contribute to age-dependent impairment of NMDA induced pial artery dilation after FPI NMDA and glutamate (10 8, 10 6 M) elicited reproducible pial small artery (120–160 Am) and arteriole (50–70 Am) dilation (Table 1). Pial small artery dilation in response to NMDA receptor activation and glutamate was reversed to vasoconstriction after FPI in the newborn pig (Fig. 2A).

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Fig. 1. Influence of FPI on CSF tPA (ng/ml) in the newborn and juvenile pig, n = 4. *P b 0.05 versus corresponding pre-FPI value. +P b 0.05 versus corresponding newborn value.

However, pretreatment with EEIIMD or suPAR (10 7 M) 30 min prior to injury, prevented NMDA and glutamate induced vasoconstriction following FPI in the newborn pig (Fig. 2A). Nonetheless, pial small artery responses to NMDA and glutamate were still impaired following FPI in the presence of EEIIMD or suPAR in the newborn pig (Fig. 2A). Similar observations were made in pial arterioles. In sham control animals not subjected to FPI, responses to NMDA and glutamate were unchanged in the presence of either EEIIMD or suPAR (data not shown). In contrast, pial small artery dilation to NMDA receptor activation and glutamate was only blunted and not reversed to vasoconstriction in the juvenile pig (Fig. 2B). Pretreatment with EEIIMD or suPAR partially restored impaired NMDA and glutamate dilation post injury in the juvenile pig (Fig. 2B). Similar observations were made in pial arterioles. On a percentage basis, the amount of protection of NMDA and glutamate dilation by EEIIMD and suPAR post injury was significantly greater in the newborn versus the juvenile pig. 3.3. EEIIMD and suPAR blunt FPI associated pial artery vasoconstriction more in newborn than juvenile pigs FPI induced pial small artery and arteriole vasoconstriction to a greater extent in newborn versus juvenile pigs (Fig. 3). However, EEIIMD or suPAR (10 7 M) pretreatment blunted injury associated pial artery vasoconstriction (Fig. 3). The protection observed with EEIIMD and suPAR was significantly greater in the newborn versus the juvenile pig (Fig. 3). 3.4. Efficacy and selectivity of EEIIMD and suPAR as plasminogen inhibitors Topical uPA and tPA (10 9, 10 7 M) elicit reproducible pial small artery and arteriole dilation (Table 1).

Fig. 2. Influence of NMDA and glutamate (10 8, 10 6 M) on pial artery diameter before (control), after FPI, after FPI in EEIIMD (10 7 M) pretreated pigs, and after FPI in suPAR (10 7 M) pretreated pigs. (A) Newborn, (B) juvenile, n = 6. *P b 0.05 versus corresponding control value, +P b 0.05 versus corresponding after FPI non pretreated value.

Fig. 3. Influence of FPI on pial small artery (SA) and arteriole (ART) diameter in the absence and presence of EEIIMD or suPAR (10 7 M) in newborn and juvenile pigs, n = 6. *P b 0.05 versus FPI alone +P b 0.05 versus corresponding value in the newborn.

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Pial artery dilation to uPA was blocked by suPAR (10 7 M) but responses to tPA were unchanged (Fig. 4A). However, pial small artery dilation to both uPA and tPA was blocked by EEIIMD (10 7 M) (Fig. 4A). In contrast, pial artery dilation to SNP and papaverine was unchanged by either EEIIMD or suPAR administration (Fig. 4B). Neither suPAR nor EEIIMD had any significant effect on pial small artery diameter by themselves (159 F 8 versus 157 F 8 for suPAR and 157 F 7 versus 154 F 9 Am for EEIIMD, respectively, n = 6). Similar observations were made in pial arterioles. 3.5. Blood chemistry and intensity of injury Blood chemistry values were obtained at the beginning and end of all experiments. These values were 7.47 F 0.03, 35 F 4, and 91 F 7 mm Hg versus 7.46 F 0.03, 34 F 3, and 90 F 8 mm Hg for pH, pCO2, and pO2, respectively, before and after injury. There were no statistical differences in these

Fig. 4. (A) Influence of uPA and tPA (10 9, 10 7 M) on pial artery diameter before (control) and after either coadministered suPAR or EEIIMD (10 7 M), n = 6. (B) Influence of SNP and papaverine (10 8, 10 6 M) on pial artery diameter before (control) and after either coadministered suPAR or EEIIMD, n = 6. *P b 0.05 versus corresponding control value.

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values between newborn and juvenile pigs. Additionally, there were no statistical differences in the blood chemistry parameters between the sham, FPI, and plasminogen activator inhibitor treated animals. The amplitude of the pressure pulse, used as an index of injury intensity, was equivalent in newborn and juvenile pigs (1.9 F 0.1 and 2.0 F 0.1 atm).

4. Discussion Results of the present study show that cortical periarachnoid CSF tPA concentration was elevated within 1 h of FPI. The CSF tPA concentration was significantly more robustly increased in the newborn versus the juvenile pig. On a molar basis, the baseline CSF concentration was approximately 10 10–10 9 M while the newborn FPI CSF concentration was approximately 10 8–10 7 M. However, the concentration present at the receptor level is unknown, but presumably somewhat greater. These data indicate that tPA is detectable in CSF under baseline conditions and that the concentration increases significantly following FPI. Since the timeframe for CSF collection was 60 min, these data suggest that the source of the tPA was that from preformed stores for plasminogen activator rather than newly synthesized tPA. However, the experimental design of the present study did not allow for the identification of the cellular site of origin for tPA detected in CSF. Potential cellular sites of origin include neurons, glia, vascular smooth muscle, and endothelial cells. Other results of this study show that pial artery dilation in response to NMDA receptor activation and glutamate was reversed to vasoconstriction following FPI in the newborn pig, consistent with previous studies [4,5,6,25,26]. Pretreatment with the putative plasminogen inhibitors EEIIMD or suPAR prevented NMDA and glutamate induced vasoconstriction post insult. However, the resulting dilator responses to these excitatory amino acids were still significantly reduced compared to that observed in the absence of brain injury. In contrast, responses to NMDA and glutamate were blunted, but not reversed to vasoconstriction, in the juvenile pig [5,6]. Pretreatment with EEIIMD or suPAR similarly partially prevented impairment of dilation to NMDA and glutamate in the juvenile pig. These data suggest that plasminogen activators contribute to age-dependent impairment of NMDA receptor mediated dilation following brain injury. Potential explanations for the age-dependent contribution of plasminogen activators to impairment of NMDA receptor mediated dilation include age related differences in the amount of plasminogen activator released following FPI (Fig. 1), the receptor expression under baseline and FPI conditions, and/or signal transduction pathway used to elicit the ultimate observed vascular effect. It is uncertain which of these possibilities is the primary explanation for the present observations.

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Additional results of this study show that the diameter of pial small arteries and arterioles is reduced in an agedependent manner following FPI, consistent with previous studies [2,5,6,3,8]. Activation of NMDA receptors contributes to age-dependent reductions in cerebral blood flow post insult [7]. New data in this study show that plasminogen activator inhibition blunted brain injury induced pial artery vasoconstriction. Furthermore, blunted pial artery vasoconstriction by the plasminogen inhibitors was age-dependent. Therefore, these data suggest that plasminogen activators contribute to age-dependent effects of FPI on pial artery diameter. Other experiments in this study were directed at characterizing the efficacy and selectivity of the plasminogen inhibitors EEIIMD and suPAR. For example, suPAR blocked uPA induced pial artery dilation while responses to tPA were unchanged. Conversely, EEIIMD blocked vasodilator responses to both uPA and tPA. Such observations indicate that suPAR, at the concentration of 10 7 M, is an efficacious inhibitor of uPA, while EEIIMD efficaciously inhibited both uPA and tPA vascular responses. The agonist concentrations used in these studies were based on the determination of CSF tPA concentration and therefore reflected the CSF concentrations observed under physiologic (baseline, uninjured) (10 9 M) and pathologic (post FPI) (10 7 M) conditions. Since vasodilator responses to the nitric oxide (NO) released SNP and the non specific dilator papaverine were unchanged by either suPAR or EEIIMD, these data further suggest that these plasminogen inhibitors are also selective in their action in the pig pial vasculature in the concentrations used in this study. Because topical administration of suPAR and EEIIMD had no significant effect on pial artery diameter in sham control animals, these data suggest that plasminogen activators have minimal effect on resting cerebrovascular tone. Additionally, plasminogen activators appear to have minimal tonic influence on vascular responses to excitatory amino acids as dilation to NMDA and glutamate was unchanged in the presence of EEIIMD or suPAR under sham (non brain injury) conditions. EEIIMD inhibited the vascular activity of uPA similar to PAI-1, but EEIIMD does not modulate tPA or UPA catalytic activity [31]. These results exclude the possibility that EEIIMD could signal by itself as had been suggested recently for PAI-1, but affirm that its effect is mediated through a non-catalytic effect of tPA and uPA on vascular tone [16]. Since effects of plasminogen inhibitors were similar in pial small arteries and arterioles, these data suggest that there are minimal regional vascular differences in the contribution of plasminogen activators to impaired NMDA receptor mediated vasodilation post brain injury. Importantly, because EEIIMD and suPAR achieved similar protection of responses to excitatory amino acids after FPI, the above selectivity data suggest that, while both tPA and uPA may contribute to impaired responses to NMDA and glutamate post insult, there appears to be a relatively greater role for endogenous uPA versus tPA in such impairment.

Pial artery vasoconstriction following brain injury could result from active vasoconstriction or withdrawal of a dilator influence. Previous studies have supported the former case in that enhanced release of endothelin-1 and vasopressin after FPI contributed to post insult reductions in pial artery diameter [5,6]. tPA is a cerebrovasodilator [14] and recent data show that pial artery dilation to exogenously administered tPA and uPA is blunted after FPI due to altered NO function [9], indicating a withdrawal of an active dilatory influence on the cerebral circulation. However, the concentration of endogenous plasminogen activator at the receptor level following FPI is unknown. Since it has been observed that uPA elicits dilation at low but constriction at higher concentrations [20], it is conceivable that endogenous plasminogen activators may produce pial artery vasoconstriction following brain injury. The observation that the plasminogen activator inhibitors EEIIMD and suPAR blunt pial artery vasoconstriction after FPI suggests that brain injury may produce both a withdrawal of a tonic dilator influence as well as actively promote plasminogen activator induced vasoconstriction. These observations also suggest that removal of a tonic dilator influence and elicitation of a vasoconstrictor may physiologically oppose NMDA receptor mediated dilation. Clinically, coagulopathy has been suggested to contribute to outcome in pediatric abusive head trauma [22]. Yet, in basic science studies, tPA has been observed to contribute to excitotoxic neuronal death by the activation of microglia [36,37] or enhancement of NMDA receptor mediated signaling [33]. For example, mice deficient in tPA were observed to be resistant to the neuronal toxic effects of the intrahippocampal injection of glutamate [37], which implicate tPA in the neurotoxic cascade of this excitatory amino acid. These data were strengthened by the observation that the administration of tPA increased the volume of injured tissue in mice subjected to stroke [39]. Furthermore, deficiency of plasminogen, the substrate for tPA and infusion of a2 antiplasmin, the endogenous plasmin inhibitor, protect mice against excitotoxin induced neuronal death [38]. Important to these studies was the observation that tPA can bind to two types of receptors in the CNS, low density lipoprotein related (LRP) [12,31] and the NR-1 subunit of NMDA [39]. The interaction between tPA and the NR-1 subunit of NMDA is prevented by PAI-1, a protein that blocks the catalytic site of tPA [33], but may also affect its interaction with LRP [32]. The implication of the interaction between tPA and the catalytic site of the NR-1 subunit of NMDA, then, relates to the observed ability of tPA to potentiate the excitotoxic consequences of NMDA receptor activation [33]. Similarly, tPA protein was abundantly expressed after spinal cord injury while there was preservation of white matter and hindlimb function in tPA deficient mice [1]. On the other hand, though, a neuroprotective effect of tPA in zinc induced neuronal death has also been documented [24]. Thus, tPA must act through more than one mechanism.

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On the basis of interspecies extrapolation of brain growth curves [17], the age period of newborn pigs chosen in the present study may approximate the newborn to infant time period in the human. Correspondingly, the age period for the juvenile pig chosen in the present study may correlate to that of a human child 8–10 years of age [17]. Although the amplitude of the pressure pulse, which reflects the intensity of the injury, was equivalent in newborn and juvenile pigs, how this force acts once it enters the skull may well depend on differences in the composition and compliance of the newborn and juvenile brain. Additionally, it is unclear how developmental parameters such as brain water content, skull dimensions, or suture elasticity will affect the biomechanics of the fluid wave pulse delivered to the brains of these two age groups. In conclusion, results of the present study show that EEIIMD and suPAR partially prevented FPI induced alterations in NMDA dilation and reductions in pial artery diameter. EEIIMD and suPAR appear to be efficacious and selective inhibitors of plasminogen activator induced dilation. Because suPAR and EEIIMD do not inhibit the plasminogen activator activity of uPA, this is likely a receptor-dependent non-proteolytic process. These data suggest that plasminogen activators contribute to agedependent impairment of NMDA induced dilation following FPI.

Acknowledgments The authors thank Antonio Pedulla for excellent technical assistance in the performance of the experiments. This research was supported by grants from the National Institutes of Health.

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